1. Field of the Invention
The field of this invention is the control of nematodes, in particular the control of soybean cyst nematodes. The invention also relates to the introduction of genetic material into plants that are susceptible to nematodes in order to increase tolerance to nematodes.
2. Background Art
Nematodes are microscopic wormlike animals that feed on the roots, leaves, and stems of more than 2,000 vegetables, fruits, and ornamental plants causing an estimated $100 billion crop loss worldwide. One common type of nematode is the root-knot nematode, whose feeding causes the characteristic galls on roots. Other root-feeding nematodes are the cyst- and lesion-types, which are more host specific.
Nematodes are present throughout the United States, but are mostly a problem in warm, humid areas of the South and West, and in sandy soils. Soybean cyst nematode (SCN), Heterodera glycines, was first discovered in the United States in North Carolina in 1954. It is the most serious pest of soybean plants. Some areas are so heavily infested by SCN that soybean production is no longer economically possible without control measures. Although soybean is the major economic crop attacked by SCN, SCN parasitizes some fifty hosts in total, including field crops, vegetables, ornamentals, and weeds.
Signs of nematode damage include stunting and yellowing of leaves, and wilting of the plants during hot periods. However, nematodes, including SCN, can cause significant yield loss without obvious above-ground symptoms. In addition, roots infected with SCN are dwarfed or stunted. SCN can decrease the number of nitrogen-fixing nodules on the roots, and may make the roots more susceptible to attacks by other soil-borne plant pathogens.
The SCN life cycle has three major stages: egg, juvenile, and adult. The life cycle can be completed in 24 to 30 days under optimum conditions. When temperature and moisture levels become adequate in the spring, worm-shaped juveniles hatch from eggs in the soil. These juveniles are the only life stage of the nematode that can infect soybean roots.
After penetrating the soybean roots, juveniles move through the root until they contact vascular tissue, where they stop and start to feed. The nematode injects secretions that modify certain root cells and transform them into specialized feeding sites. The root cells are morphologically transformed into large multinucleate syncytia or giant cells, which are used as a source of nutrients for the nematodes. The actively feeding nematodes thus steal essential nutrients from the plant resulting in yield loss. As the nematodes feed, they swell and eventually female nematodes become so large that they break through the root tissue and are exposed on the surface of the root.
Male nematodes, which are not swollen as adults, migrate out of the root into the soil and fertilize the lemon-shaped adult females. The males then die, while the females remain attached to the root system and continue to feed. The swollen females begin producing eggs, initially in a mass or egg sac outside the body, then later within the body cavity. Eventually the entire body cavity of the adult female is filled with eggs, and the female nematode dies. It is the egg-filled body of the dead female that is referred to as the cyst. Cysts eventually dislodge and are may be found free in the soil. The walls of the cyst become very tough, providing excellent protection for the 200 to 400 eggs contained within. SCN eggs survive within the cyst until proper hatching conditions occur. Although many of the eggs may hatch within the first year, many also will survive within the cysts for several years.
SCN can move through the soil only a few inches per year on its own power. However, SCN can be spread substantial distances in a variety of ways. Anything that can move infested soil is capable of spreading SCN, including farm machinery, vehicles and tools, wind, water, animals, and farm workers. Seed sized particles of soil often contaminate harvested seed. Consequently, SCN can be spread when seed from infested fields is planted in non-infested fields. There is even evidence that SCN can be spread by birds. Only some of these causes can be prevented.
Traditional practices for managing SCN include: maintaining proper fertility and soil pH levels in SCN-infested land; controlling other plant diseases, as well as insect and weed pests; using sanitation practices such as plowing, planting, and cultivating of SCN-infested fields only after working non-infested fields; cleaning equipment thoroughly with high pressure water or steam after working in infested fields; not using seed grown on infested land for planting non-infested fields unless the seed has been properly cleaned; rotating infested fields and alternating host crops with non-host crops, such as, corn, oat and alfalfa; using nematicides; and planting resistant soybean varieties.
Methods have been proposed for the genetic transformation of plants in order to confer increased resistance to plant parasitic nematodes. U.S. Pat. Nos. 5,589,622 and 5,824,876 are directed to the identification of plant genes expressed specifically in or adjacent to the feeding site of the plant after attachment by the nematode. The promoters of these plant target genes can then be used to direct the specific expression of toxic proteins or enzymes, or the expression of antisense RNA to the target gene or to general cellular genes. The plant promoters may also be used to confer cyst nematode resistance specifically at the feeding site by transforming the plant with a construct comprising the promoter of the plant target gene linked to a gene whose product induces lethality in the nematode after ingestion. However, these patents do not provide any specific nematode genes that are useful for conferring resistance to nematode infection, and the methods are only useful for expressing genes specifically at the feeding sites for nematodes after attachment to the plant.
Recently, RNA interference (RNAi), also referred to as gene silencing, has been proposed as a method for controlling nematodes. When double-stranded RNA (dsRNA) corresponding essentially to the sequence of a target gene or mRNA is introduced into a cell, expression from the target gene is inhibited (See e.g., U.S. Pat. No. 6,506,559). U.S. Pat. No. 6,506,559 demonstrates the effectiveness of RNAi against known genes in C. elegans, but does not teach or suggest any novel genes that are essential for plant parasitic nematodes, and does not demonstrate the usefulness of RNAi for controlling plant parasitic nematodes.
In addition, RNAi was used in PCT Publication WO 01/96584 to target nematode genes, preferably in root-knot nematodes and potato cyst nematodes. Preferred targets included molecules involved in ribosome assembly; neurotransmitter receptors and ligands; electron transport proteins; metabolic pathway proteins; and proteins involved in protein and polynucleotide production, folding, and processing. However, none of the sequences provided in PCT Publication WO 01/96584 were demonstrated to be down-regulated using RNAi, and moreover, they were not shown to be useful in conferring resistance to plant parasitic nematodes.
PCT Publication WO 01/17654 A2 also proposed the use of RNAi for targeting essential plant pathogenic and parasitic nematode genes. The host plant is preferably transformed with a construct for expressing dsRNA that has substantial sequence identity to an endogenous and essential nematode gene. The publication proposes that the invention is particularly useful for targeting a vascular acetylcholine transporter protein, a choline acetyltransferase, and a ubiquinone oxidoreductase. WO 01/17654 demonstrated that RNAi was effective in reducing expression of sec-1, involved in vesicle trafficking, in Meloidogyne incognita. However, sec-1 was not shown to be essential for plant parasitic nematodes or useful for conferring plant resistance to nematodes. In addition, the patent publication does not teach or suggest any novel genes that are essential for plant parasitic nematodes.
A number of models have been proposed for the action of RNAi. See, e.g., Hammond et al. (2001) Nature Reviews Genetics 2, 110-119, and references cited therein. In mammalian systems, dsRNAs larger than 30 nucleotides trigger induction of interferon synthesis and a global shut-down of protein syntheses, in a non-sequence-specific manner. See, e.g., Bass (2001) Nature 411, 428-429; Elbashir, et al. (2001) Nature 411, 494-498. However, U.S. Pat. No. 6,506,559 discloses that in nematodes, the length of the dsRNA corresponding to the target gene sequence may be at least 25, 50, 100, 200, 300 or 400 bases, and that even larger dsRNAs (742 nucleotides, 1033 nucleotides, 785 nucleotides, 531 nucleotides, 576 nucleotides, 651 nucleotides, 1015 nucleotides, 1033 nucleotides 730 nucleotides, 830 nucleotides, see Table 1) were also effective at inducing RNAi in C. elegans. Moreover, Wesley, et al. (2001) The Plant Journal 27, 581-590 discloses that when hairpin RNA constructs having double stranded regions ranging from 98 to 854 nucleotides were transformed into a number of plant species, the target plant genes were efficiently silenced. There is general agreement that in many organisms, including nematodes and plants, large pieces of dsRNA are cleaved into 21-23 nucleotide fragments (siRNA) within cells, and that these siRNAs are the actual mediators of the RNAi phenomenon.
Notwithstanding the foregoing, there is a need to identify safe and effective compositions and methods for the controlling plant parasitic nematodes using RNAi, and for the production of plants having increased resistance to plant parasitic nematodes.